1. Field of the Invention
The present invention relates to a voltammetric sequence-selective sensor for target polynucleotide sequences, and more particularly to a polynucleotide probe having one end immobilized on an amperometric electrode and a target binding region.
2. Description of Prior Art
The application of recombinant DNA techniques is emerging as a powerful tool in the area of molecular diagnostic medicine. For example, the development of DNA and RNA molecular probes for the detection of viral and bacterial genomes and genetic defects in mammalian chromosomes may replace current immunochemical approaches.
Polynucleotide hybridization assays are used as research tools for the detection and identification of unique or specific polynucleotide sequences in samples of complete, fragmented, or mixed nucleic acids. Various hybridization diagnostic techniques have been developed.
The southern blot technique is based on a polynucleotide hybridization technique employing radiolabeled nucleic acid probes. This procedure permits autoradiographic detection of probe/analyte hybrids and identification of the polynucleotide sequence of the analyte. However, the Southern procedure, as well as the other diagnostic procedures employing radiolabeled nucleic acid probes, are very complex, time consuming, and have the additional problems and expenses generally associated with radioactive materials such as disposal and personnel monitoring. Thus, such assays have remained a tool of basic research and are not generally employed in applied or commercial areas such as clinical diagnosis.
Most of the existing methods used to attach a polynucleotide probe to a solid support are non-specific and the number of attachment sites per nucleic acid is difficult to control. It has been found that multiple attachment reduces the degree of freedom of the immobilized nucleic acid. The physical adsorption of single stranded DNA, covalent attachment via diazo-linkage, epoxidation, cyanogen bromide activation and photochemical reactions are associated with the complication of non-specific linkage between the nucleic acids and the solid support.
Canadian Patent 1,223,222, issued on Jun. 23, 1987, discloses an immobilized nucleic acid-containing probe coupled to a solid support in a manner which is site specific, which does not interfere with the ability of the nucleic acid to hybridize and which involves preferably a single chemical covalent linkage per nucleic acid to the solid support. Specifically, the nucleotide is coupled to the nucleic acid employing an enzyme and the nucleotide is chemically modified.
Canadian Patent 1,236,410, issued on May 10, 1988, discloses methods and reagents for determining the presence of specific DNA and RNA base sequences on single stranded target polynucleotides. The method involves the preparation of a specific single stranded ribonucleic acid or deoxyribonucleic acid molecule into which a bifunctional crosslinking molecule has been covalently incorporated. The incorporation is such that the crosslinking molecule retains the capacity to undergo a second addition to the nucleic acid of the bacterial, viral, or mammalian chromosome which is the target for the probe. The single stranded DNA or RNA probe is designed so that its nucleic acid base sequence is complementary to a unique region of the bacterial, viral, or mammalian chromosome target sequence. The nucleic acid, for example, from a blood, tissue, or cell sample is reacted with the probe under conditions where hybridization of the probe with the target will occur. Following hybridization, the sample is subjected to a photochemical or chemical procedure which causes crosslinking of the probe to the target complementary sequence. If no target genomic sequence is present, then no crosslinking of the probe will occur. In some cases hybridization of the probe to the target will precede both reactions of the bifunctional crosslinking reagent.
Canadian Patent 1,293,937 issued on Jan. 7, 1992, discloses polynucleotide probe compositions, diagnostic kits, and nonradiometric hybridization assays useful in the detection and identification of at least one target polynucleotide analyte in a physiological sample. There is provided a first polynucleotide probe having a catalyst attached thereto and which is substantially complementary to a first single-stranded region of the analyte; and a second polynucleotide probe having an apoluminescer attached thereto and which is substantially complementary to a second single-stranded region of the analyte. The second region is substantially mutually exclusive from the first region, such that upon hybridization of the first and second probes with the analyte, the catalyst and the apoluminescer are close enough to each other to permit the catalyst to act on a substrate to release a transformation radical to convert the apoluminescer to a luminescer.
U.S. Pat. No. 4,882,013 issued on Nov. 21, 1989, discloses the use of tetrathiafulvalene (TTF) and its derivatives as mediator molecules in the transfer of electrons between redox systems and electrodes in bioelectrochemical processes. There is also disclosed an assay procedure for assaying a substance based on this redox system and using an electrode. This assay does not include the detection of hybridized polynucleotide probes with a target polynucleotide since no redox system is involved in this hybridization.
Current methods for the diagnosis of inherited diseases employ digestion of a prepared DNA sample with restriction enzymes to form short, double-stranded segments, gel electrophoresis to separate these segments according to size, transfer of the separated segments to a thin membrane material, such as nylon, hybridization of the segments of interest with a labeled oligonucleotide (of complementary sequence to the known disease sequence), and detection of the label. The complete procedure requires about 24 hours, is labor-intensive, and is not readily automated. Furthermore, these methods usually employ radioactive labels, with their inherent safety and disposal problems. None of the above-mentioned diagnostic systems discloses a probe which can be treated to be reusable for hybridization. Thus, these systems are for a unique usage.
None of the above-mentioned diagnostic systems are highly sequence specific to enable the diagnosis of point mutation inherited diseases.
It would be highly desirable to be provided with a sequence-selective system for target polynucleotide sequences that uses a nonradiometric label in a system that is simple to use, highly specific and sensitive and reusable.